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Grape and wine analysis: Oenologists to exploit advanced test kits.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

The present study, taraxerone (D-friedoolean-14-en-3-one) was isolated from Sedum sarmentosum with purity 96.383%, and its enhancing effects on alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were determined: EC50 values were 512.42 ± 3.12 and 500.16 ± 3.23 μM for ADH and ALDH, respectively. In order to obtain more information on taraxerone related with the alcohol metabolism, 40% ethanol (5 mL/kg body weight) with 0.5–1 mM of taraxerone were administered to mice. The plasma alcohol and acetaldehyde concentrations of taraxerone-treated groups were significantly lowered than those of the control group (p < 0.01): approximately 20–67% and 7–57% lowered for plasma alcohol and acetaldehyde, respectively. Compare to the control group, the ADH and ALDH expressions in the liver tissues were abruptly increased in the taraxerone-treated groups after ethanol exposure. In addition, taraxerone prevented catalase, superoxide dismutase, and reduced glutathione concentrations from the decrease induced by ethanol administration with the concentration dependent manner.

Effects of Chitooligosaccharide Lactate Salt on Activity of Acetaldehyde Dehydrogenase.

Chitooligosaccharides (COS), a kind of oligosaccharide made from chitin or chitosan, have been used a popular remedy for hangovers. In this study we investigated the in vitro effect of COS lactate salt on ethanol-induced cytotoxicity and the in vitro effect of short-term COS lactate salt feeding on ethanol-induced hangover. Pretreatment of HepG2 cells with COS lactate salt significantly reduced ethanol-induced cytotoxicity and suppressed generation of reactive oxygen species. In addition, COS lactate salt dose-dependently increased acetaldehyde dehydrogenase (ALDH) activity in vitro and reversed the ALDH inhibition induced by daidzin. Furthermore, oral administration of COS lactate salt (200 mg/kg) for 5 days significantly decreased the blood levels of alcohol and acetaldehyde in ethanol-treated mice. It was also demonstrated that hepatic mitochondrial ALDH activity was significantly increased in COS lactate salt-treated mice. Taken together, these findings indicate that COS lactate salt may have efficacy for the management of alcoholic hangovers.

Exploitation of the semi‐homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies.

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATαho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4 -disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.

Potential of the waste from beer fermentation broth for bio-ethanol production without any additional enzyme, microbial cells and carbohydrates.

The potential of the waste from beer fermentation broth (WBFB) for the production of bio-ethanol using a simultaneous saccharification and fermentation process without any extra additions of saccharification enzymes, microbial cells or carbohydrate was tested. The major microbial cells in WBFB were isolated and identified. The variations in compositions of WBFB with stock time were investigated. There was residual activity of starch hydrolyzing enzymes in WBFB. The effects of reaction modes, e.g. static and shaking on bio-ethanol production were studied. After 7 days of cultivation using the supernatant of WBFB at 30°C the ethanol concentration reached 103.8 g/L in shaking culture and 91.5 g/L in static culture. Agitation experiments conducted at a temperature-profile process in which temperature was increased from 25 to 67°C shortened the simultaneous process time. The original WBFB was more useful than the supernatant of WBFB in getting the higher concentration of ethanol and reducing the fermentation time. From this whole study it was found that WBFB is a cheap and suitable source for bio-ethanol production.

Control of volatile acidity (VA) is a major issue for wine quality. In this study, we investigated the production of VA by a deletion mutant of the fermentation stress response gene AAF1 in the budding yeast Saccharomyces cerevisiae. Fermentations were carried out in commercial Chardonnay grape must to mimic industrial wine-making conditions. We demonstrated that a wine yeast strain deleted for AAF1 reduced acetic acid levels in wine by up to 39.2% without increasing the acetaldehyde levels, revealing a potential for industrial application. Deletion of the cytosolic aldehyde dehydrogenase gene ALD6 also reduced acetic acid levels dramatically, but increased the acetaldehyde levels by 41.4%, which is not desired by the wine industry. By comparison, ALD4 and the AAF1 paralog RSF2 had no effects on acetic acid production in wine. Deletion of AAF1 was detrimental to the growth of ald6Δ and ald4Δald6Δ mutants, but had no effect on acetic acid production. Overexpression of
AAF1 dramatically increased acetic acid levels in wine in an Ald6p-dependent manner, indicating that Aaf1p regulates acetic acid production mainly via Ald6p. Overexpression of
AAF1 in an ald4Δald6Δ strain produced significantly more acetic acid in wine than the ald4Δald6Δ mutant, suggesting that Aaf1p may also regulate acetic acid synthesis independently of Ald4p and Ald6p.

Background: We recently reported that exposure of human cells in vitro to acetaldehyde resulted in activation of the Fanconi anemia-breast cancer associated (FA-BRCA) DNA damage response network. Methods: To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA-BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase 1B. Results: Incubation of HeLa-ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media which was prevented by co-incubation with 4-methyl pyrazole (4-MP), a specific inhibitor of ADH. Ethanol treatment of HeLa-ADH1B cells produced a 4-fold increase in the acetaldehyde-DNA adduct, N2-ethylidene-dGuo, and also resulted in activation of the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network, as indicated by a monoubiquitination of FANCD2, and phosphorylation of BRCA1. Ser 1524 was identified as one site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4-MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all three responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions: Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage which activates the FA-BRCA DNA damage response network.

Effect of thermal processing during yogurt production upon the detection of staphylococcal enterotoxin B.

This research was conducted to examine the inherent properties of yogurt contaminated with staphylococcal enterotoxin B (SEB). Two types of yogurts were produced for this study. Type I yogurts were produced by adding SEB at the start of yogurt production, and type II yogurts were produced by adding SEB after the milk base had been boiled. Biochemical characteristics inherent to yogurt, including pH, lactic acid and acetaldehyde concentrations, were analyzed weekly for each batch beginning at a time just after production and throughout a storage period of at least 4 weeks. The presence of toxin during yogurt production did not result in any significant biochemical or physical changes in yogurt. However, we were unable to detect SEB toxin in type I yogurt using a commercially available enzyme-linked immunosorbent assay (ELISA). In contrast, SEB was easily detectable by our ELISA in type II yogurt samples. Higher levels of SEB were recovered from type II yogurt that had been stored for 1 week than from type II yogurt that had been stored for any other length of time. These results indicate that the biochemical characteristics of yogurt did not change significantly (relative to control yogurt) in the presence of either thermally processed SEB or native SEB. However, the ability to detect SEB by ELISA was dependent on whether the toxin had been processed.